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Our project uses novel methods for epigenomic profiling that detect 3D contact sites without cross-linking and with much higher resolution than current technologies. We recently introduced a novel strategy for chromatin profiling called CUT&RUN (Cleavage Under Targets & Release Using Nuclease), in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. The method yields precise transcription factor (TF) profiles, yet is simple to perform and is inherently robust, with extremely low backgrounds requiring ~1/10 th the sequencing depth of chromatin immunoprecipitation (ChIP). CUT&RUN binding and cleavage occurs in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the 3D chromatin environment. Together with our new native ChIP-seq protocol, we distinguish direct \u201canchor\u201d sites of a \nchromatin-bound protein from contacting sites without fixation, a kind of inference has not been possible with current methods of interrogating 3D chromatin architecture. We have two Aims: First, we will annotate the genome at high density for CTCF and cohesin sites, distinguishing between anchor and contact sites in 4D Nucleome cell lines. Second, we will continue development of a new replacement technology for 3D contact mapping, using CUT&RUN as a \u201cfront-end\u201d for proximity ligation (CUT&PASTE \u2013 Cleave Under Targets & Polish And Splice Touching Ends), building on our novel sci-HiC protocol for high-resolution TF-specific 3D interaction mapping. By annotating more contact sites in genomes and assigning the directionality of contacts, we move towards a mechanistic model of how topology within the nucleus is organized. 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